Analysis of biological specimens is a vital and critical technique for both clinical and research applications. However, depending on the source material and processing methods, it is often impossible to analyze specimens immediately after procurement. Thus, various techniques for fixation and stabilization of biological specimens have been developed.
These methods all strive to maintain antigenic sites of interest while still providing for robust preservation of the biological source material. Of the many different types of techniques that have been described in the literature, most of these utilize various chemical compounds that preserve antigenic sites through chemical cross-linking. Aldehydes are some of the most commonly used chemical fixatives. It has been shown in the literature that aldehyde-fixation occurs through a Schiff acid-base reaction resulting from the formation of covalent bonds between adjacent amine-containing groups. Formaldehyde is one of the most commonly used aldehydes for fixation. Formalin is a commercial preparation of formaldehyde in solution.
Despite the wide use of formaldehyde in fixation techniques, there are drawbacks to its use for some applications. Formaldehyde fixation actually occurs through two distinct kinetic phases. At neutral pH, it penetrates the cell, modifies primary amines such as lysine, thiols such as cysteine, as well as purine nucleic acid bases. At higher pH, the cross-linking process occurs more rapidly. However, over extended time, formaldehyde continues to react with methylol derivatives. These secondary reactions can result in the eventual degradation of antigenic sites and the native light scatter properties of the cells may be modified. Thus, it may be necessary to remove the formaldehyde after the desired level of fixation has been achieved. The fixation time is generally an empirical measure that must be optimized depending on the type of specimen and antigenic determinants. This represents a significant drawback.